This project has three interrelated objectives: (1) to provide structural information for the function of major voltage-gated potassium (Kv) channels in the heart, (2) to understand why mutations in Kv channel components lead to loss- or gain-of-function, and (3) to identify novel therapeutic strategies targeting cardiac Kv channels. The focus of this proposal is the slow delayed rectifier (IKs) channel. IKs has 2 major components: pore-forming KCNQ1 channel and auxiliary KCNE1 subunits. In human ventricles, IKs functions as a 'repolarization reserve': in response to b-adrenergic stimulation IKs increases its current amplitude to prevent excessive prolongation of action potential duration (APD). In human atria, IKs may be a liability factor for atrial fibrillation (AF). Eight 'gain-of-function' KCNQ1 mutations have been identified that are linked to familial AF. More importantly, KCNE1 upregulation has been reported for acquired AF due to valvular diseases, suggesting an increase in IKs under these conditions that can contribute to APD shortening and AF perpetuation. We have shown that another KCNE subunit expressed in human heart, KCNE2, is colocalized with KCNQ1 & KCNE1 in adult cardiac myocytes. It can associate with the IKs channel to form a KCNQ1/KCNE1/KCNE2 ternary complex. KCNE2 reduces the IKs current amplitude without affecting its gating kinetics. The importance of KCNE2 as a IKs regulator is highlighted by the identification of a familial AF-related mutation, R27C that negates the current suppressing effect of KCNE2 on IKs. The relationship between KCNE1 & KCNE2 in terms of their regulation of the IKs current amplitude in the heart is not clear. Nor is the mechanism(s) underlying their distinctly different effects on the KCNQ1 channel function. This project is designed to address these issues. We are 3 research groups with complementary expertise (Tseng - channel biophysics, Cui - computational modeling, and Tian - NMR) making a concerted effort to accomplish the following Specific Aims. Aim 1 is to determine the packing and gating-associated movements of transmembrane helices (TMHs) in the KCNQ1 channel. Aim 2 is to determine the impact of KCNE1 association on the TMH interactions in the KCNQ1 channel, and the contacts between KCNE1 & KCNQ1. Aim 3 is to determine the contacts between KCNE2 & KCNQ1 and the state-dependence of such contacts. Spatial relationships determined in these experiments will be used to constrain KCNQ1 homology models in open & closed states. We will also dock the KCNE NMR structures, after refinement, to the KCNQ1 homology models in a manner consistent with experimental data. Finally, we will test whether membrane permeable KCNE2-mimetic peptides can disrupt KCNQ1/KCNE2 interactions and increase the IKs current amplitude in cardiac myocytes (Aim 4). This could provide insights into the relationship between KCNE1 & KCNE2 in terms of IKs amplitude regulation. It also serves as a prototype for therapeutic peptides targeting KCNQ1/KCNE interactions.